Development of a hydrolysis probe-based real-time assay for the detection of tropical strains of Fusarium oxysporum f. sp. cubense race 4

Abstract

Fusarium oxysporum f. sp. cubense (Foc) is one of the most important threats to global banana production. Strategies to control the pathogen are lacking, with plant resistance offering the only long-term solution, if sources of resistance are available. Prevention of introduction of Foc into disease-free areas thus remains a key strategy to continue sustainable banana production. In recent years, strains of Foc affecting Cavendish bananas have destroyed plantations in a number of countries in Asia and in the Middle East, and one African country. One vegetative compatibility group (VCG), 01213/16, is considered the major threat to bananas in tropical and subtropical climatic conditions. However, other genetically related VCGs, such as 0121, may potentially jeopardize banana cultures if they were introduced into disease-free areas. To prevent the introduction of these VCGs into disease-free Cavendish banana-growing countries, a real-time PCR test was developed to accurately detect both VCGs. A previously described putative virulence gene was used to develop a specific combination of hydrolysis probe/primers for the detection of tropical Foc race 4 strains. The real-time PCR parameters were optimized by following a statistical approach relying on orthogonal arrays and the Taguchi method in an attempt to enhance sensitivity and ensure high specificity of the assay. This study also assessed critical performance criteria, such as repeatability, reproducibility, robustness, and specificity, with a large including set of 136 F. oxysporum isolates, including 73 Foc pathogenic strains representing 24 VCGs. The validation data demonstrated that the new assay could be used for regulatory testing applications on banana plant material and can contribute to preventing the introduction and spread of Foc strains affecting Cavendish bananas in the tropics.

Fig 1. FWB-TR4 qPCR standard curve built with a 10-fold serial dilution of plasmid DNA positive control diluted in water (grey line) or in a background of banana DNA (black line).

Fig 2. Parametrical comparison between samples pre-treatments before PCR.

Comparisons were performed testing the type of sample (dry or frozen banana tissue) and the dilution of the DNA after extraction (raw DNA diluted 10 times or raw DNA diluted 100 times).

Fig 3. Decision flowchart for PCR detection of tropical strains of Foc race 4 using FWB- TR4 and 18S uni F/R/P primers.